Our laboratory recently discovered that blue light photoactivation of insect Cryptochrome (Cry) cause rapid membrane depolarization and up to 300% increased action potential firing rate over baseline dark spontaneous firing in central brain neurons (Sheeba et al., 2007; Fogle et al., 2011). The electrophysiological light response is robust in the absence of all opsin-based classical photoreceptor inputs (Fogle et al., 2011). Genetically targeted expression of Cry in normally light-insensitive olfactory neurons confers electrophysiological light responsiveness, indicating that Cry expression may be used for optogenetic applications (Fogle et al., 2011). A combination of molecular-genetic and pharmacological experiments indicate that Cry's light sensitivity is mediated through light-activated changes in the redox state of the flavin adenine dinucleotide (FAD) chromophore bound to dCry which then couples to a redox sensor in cytoplasmic potassium channel subunits and modulate potassium channel activity. We propose to extend these findings by determining the precise molecular mechanism of how light activated Cry undergoes an intramolecular transfer of redox state from the flavin chromophore to the protein surface of Cry by testing mutants which lack a well conserved tri-tryptophan motif characterized in other Cry proteins as conducting redox signals. We will then test the hypothesis that redox transfer takes place to target proteins in the membrane. Based on strong preliminary data that membrane coupling of Cry's light activated redox state occurs through voltage gated potassium channels, we will test the hypothesis that dCry then interacts with membrane redox-sensitive effector Hyperkinetic beta subunit (Hk) of voltage-gated potassium (Kv) channels. Our preliminary data indicates that light activation of Cry rapidly modulates cellular potassium currents and depolarizes the membrane potential. We have begun testing this hypothesis and find that the lLNv electrophysiological light response in almost completely abolished in Hk null mutant flies, suggesting that Hk is the primary membrane target for the novel dCry-based phototransduction mechanism. We will determine whether rapid translocation of dCry to the neuronal membrane increases the speed and the amplitude of the electrophysiological light response, as tested using a chemical biology-based inducible strategy. These experiments provide a unique opportunity to unravel a novel non-opsin phototransduction mechanism based on redox sensing. We have also the first opportunity to examine real-time actions of Cry in vivo and the possibility of determining a biological function for the highly conserved redox sensor in KvBeta subunits. As Cry's chromophore, FAD, is the ubiquitously expressed, our work may provide the basis of a new Vitamin B-based optogenetic technology applicable to cells that do not synthesize adequate levels of retinal.